1. Field of the Invention
This invention concerns a novel and improved process, including novel preservation solutions, for long-term preservation of organs for transplantation. The preservation process comprises perfusing the organs such as liver, kidney, pancreas, spleen, brain, embryo, testicles, ovaries, lung or heart-lung complex, or washing organs such as cornea, skin or cartilage, with a first novel physiological preservation solution containing pyruvate, under normal physiological conditions and at a warm temperature to remove blood and other impurities and debris by the increased flow through the organ, bringing the organ to its basal metabolic rate stage with a second preservation solution containing pyruvate and a small percentage of alcohol and preserving the organ with diffusion of gases and nutrients from the media, by submerging and storing the organ in the first preservation solution at low but not freezing temperature for periods longer than 24 hours.
2. Related Disclosures
Organ transplantations, in particular transplantations of organs such as liver, kidney, lung, lung-heart complex, spleen, brain, testicles, embryo and skin, lens and cartilage have become an important tools in saving lives in patients with irreversibly diseased or damaged organs. With increasing incidence of the circulatory, cancerous, hereditary or infectious diseases in the population, the organ transplantation is used more and more to preserve a life of otherwise healthy individuals with badly damaged organs due to such diseases, infections, tumors and the hereditary or other conditions. Consequently, a demand for organs suitable for transplantation has risen substantially.
There are certain primary requirements for the organ to be suitable for transplantation First, the organ must be healthy. Second, it must be transplanted or transplantable in certain time in which it is possible to preserve its normal physiological function. Third, it must be immunologically acceptable to the organ's recipient.
The first requirement can be only met by a physician removing the organ from the donor's body. The third requirement is increasingly being made possible by improved understanding of immune mechanism and method for preventing organ rejection by the recipient's immune system. The pharmaceutical industry is constantly developing and designing new immunosuppressant drugs which allow the easier immunological matching of the donor and the recipient and prevent, as much as possible, the organ rejection by recipient. Drugs such as azathioprine, monoclonal antibody muromonab-CD3, cyclophosphamide, cyclosporine and other recently discovered drugs such a for example drug known as FK-506 now allow the suppression of immune reactions for up to 6 months, at which time the body of the recipient is able to substantially rebuild proteins in the transplanted organ with their own proteins thus making it more immunologically acceptable. Moreover, when the graft tissue becomes accommodated within recipient's body, it can be maintained with relatively small and reasonably well tolerated doses of immunosuppressive drugs.
Consequently, the only remaining obstacle for the successful transplantation of organs is the preservation of their anatomical and functional integrity, in particular the preservation of their normal function for any length of time. With geographical spread of possible donors over the whole world, the length of time longer than 24 hours for preservation is extremely important.
There are several different circumstances involved in the organ transplantation. First, there is a group of organs which are metabolically highly active and have greater demands for supplies of oxygen and nutrients. In this group are organs such as heart, heart-lung complex, brain and embryo. The heart, brain and embryo in particular can be quickly and irreversibly damaged during extended time of ischemia caused by the insufficient supply of oxygen. These organs have to be prewashed by perfusion and brought to the basal metabolic state as soon as possible.
The second group of organs are the organs which have extensive vascularization and blood circulation and perform their function via such blood circulation by metabolizing, exchanging, releasing, detoxifying or eliminating certain compounds and gases in or out of the blood. These organs, while very active metabolically, are not as sensitive to the oxygen supply as is the first group of organs. Among these organs are liver, kidney, pancreas, spleen and lung. Since these organs contain large amounts of blood in their inner circulation, they also need to be prewashed by perfusion because the remnants of the blood and other tissue debris in their vessels may cause clotting of capillaries, and catabolic by-products such as iron, bile, aldehydes, etc., cause tissue acidity, alkalinity, or other homeostatic inequilibrium which may result in tissue damage.
The third and the least sensitive group of organs to the presence of oxygen and nutrients, used for transplantation are cartilage, cornea and skin. These organs do not have high requirement either for blood circulation or blood removal and the remnants of the blood and tissue debris may be simply washed in the oxygenated preservation solution. Their storage prior to transplantation could therefore continue for almost unlimited time if the suitable preservation solution containing oxygen and some energy supply is provided.
The ease of the organ preservation depends on their function which, in turn, determines their energy and oxygen demands.
The primary function of the heart is its continuous pumping of blood through the blood circulation system. That function depends on uninterrupted myocardial contractility which, in turn, depends on uninterrupted supply of energy and oxygen. Myocardial contractility must be preserved even during the time when the heart is removed from the donor and transplanted into the recipient. Since the contracting heart needs the constant supply of energy and oxygen, if these are not available, myocardial ischemia caused by inadequate circulation of blood to the myocardium develops which in turn results in irreversible destruction of the myocardial contractility.
The recently more often used heart-lung transplant is unique in that it requires a preservation of not only of myocardial function but also the alveoli-capillary exchange of gases. Consequently, it is important that even minuscule lung capillaries are preserved in the fully functional conditions. That requires a removal of all remnants of the blood and debris from the blood capillaries so that capillaries are not congested with the blood cells and debris, dead blood cells, blood clots or by blood catabolism by-products. The perfusion of heart-lung complex prior to preservation and storage is required.
The primary function of liver is metabolism, detoxification and the removal of the metabolic by-products and other toxic or potentially toxic materials from the blood, and redirecting these materials via blood connection to the kidney for the elimination from the body. The liver is one of the most vascularized organs in the body filled at any given time with a large amount of blood. Consequently, the liver also must be perfused before storing in the preservation solution and its metabolism must be decreased prior to transplantation.
Kidneys anatomical organization consists of extensive microcirculation interconnected with the complementary network of the excretory capillary system for elimination of catabolites, ions and water from the body. Prior to storing the kidney for transplantation, it is necessary to rinse away all blood from the renal vasculary bed. Spleen, which acts as a refuse for blood cells destruction by-products, is in the same group as kidney and liver and needs to be perfused before being transplanted. Testicles and ovaries are glandular organs which are reasonably vascularized and also would require perfusion to wash away the blood.
Because of their active function, liver, spleen and kidney have lively metabolism and may require energetic and nutritious supply. Consequently, in the interim between their removal from the donor's body and before transplanting them into a recipient's body, it is necessary to decrease their metabolism to a basal level.
Until now it has not been possible to successfully transplant the brain of one individual to another. Nevertheless, experimental designs exist and attempts are continuously made to bring such transplantation possibility about. Since the brain is the organ extremely sensitive to the deficiency of oxygen and energy supply, its preservation for transplantation, when feasible, will require a prior perfusion to remove all blood and other metabolites which could or would cross blood-brain barrier and also the use of the preservation solution with continuous perfusion which will allow the brain to exist in low metabolic state and provide at the same time essential nutrition, oxygen and energy to preserve sensitive nerve cells intact and fully functional.
Preservation of organs is currently commonly achieved by hypothermia and by perfusion with certain cardioplegic or other physiological solutions. The detailed description of the procedure for long-term preservation of the heart for transplantation is subject of concomitant and copending patent application entitled "Novel and Improved Technology for Preservation of the Heart for Transplantation", Ser. No. 07/455,580 filed Dec. 21, 1989, hereby incorporated by reference.
Surgical procedures including a removal of the organ for transplantation and inserting the organ to the donor body are individually specific to the organ but generally require a bloodless, relaxed and motionless field during operation. In the case of heart, this is currently accomplished by ischemic arrest induced by cross clamping the aorta which however causes myocardial ischemia. Any period of cardiac ischaemia and/or disrupted circulation to the organ resulting in organ ischemia is accompanied by metabolic and structural changes which determine the functional recovery of the organ in the postoperative period. The safe period of ischaemia for the human heart or for disruption of circulation in other organs and brain varies and is not clearly defined, but 20-30 minutes is generally considered to be the upper limit at least for heart, with much shorter periods for brain where even the short term oxygen deficiency may cause the irreversible damage to the nerve cells.
The need for protection of the organs during disruption of circulation has been well recognized and a number of methods including local and systemic hypothermia, intermittent perfusion, retrograde perfusion with cold blood, perfusion with cold lactated Ringer's solution, tetrodotoxin, acetylcholine, chemical asanguinous K.sup.+ cardioplegia and cold blood cardioplegia and perfusion, have all been used in experimental studies and clinical practice in the field of organ transplantations. Of these, hypothermia and pharmacological treatments with various cold carioplegic or other solutions have now gained wide acceptance in clinical practice.
Hypothermia has been proved to be an effective method of organ preservation. It provides a decrease in organ metabolism, lowers the energy requirements, delays the depletion of high energy phosphate reserves and lactic acid accumulation, and retards the morphological and functional deterioration associated with disruption of blood supply. The technique of topical cooling with continuous irrigation of the surface of the organ was first described in Surg. Gynaecol. Obst., 129:750 (1959). In this technique, the cooling proceeded from the surface of the organ to the interior but was unlikely to cool the internal organ tissue without profound hypothermia. Such moderate hypothermia and surface cooling have been generally found inadequate to protect the organ for more than one hour. Hypothermia with combined with improper solutions results in edema. Edema influences the voltage of the cells and destroys membrane potential integrity. On the other hand, deep hypothermia and surface cooling which is generally sufficient for 90 minutes from the organ removal is known to cause tissue damage due to crystallization of intracellular water and the membrane lipids. Canad. Anaesth. Soc. J., 27:381 (1980). Postgrad. Med. J., 59:11 (1983) reports that hypothermia slows all metabolic processes (thus conserving energy) including damaging degradative mechanisms and pathways which produce toxic metabolites. Hypothermia further leads to constriction and collapse of the vascular bed. The efficacy of hypothermia as a protective agent was reported as the post-ischemic recovery of function following a 60 minute period of ischemic arrest in the rat heart. In a case of the heart, reduction of the myocardial temperature from 37.degree. C. to 4.degree. C. during ischemia from resulted in a progressive improvement of post-ischemic recovery from 0% to 96% of preischemic function. The hypothermic protection is reported to be poor and falling off rapidly as the organ temperature rises above 28.degree. C. In contrast, organ preservation below 24.degree. C. temperature was reasonable for short time and was little improved with increasing degrees of hypothermia. The reason for the sharp inflection is unknown but might be related to lipoprotein phase transitions in cell membranes.
In view of the reported findings that (a) the moderate hypothermia is inadequate to protect the organ for more than one hour; (b) profound cooling of organ may cause cellular damage; and (c) that combination of appropriate protection solution with a mild hypothermia can only preserve the metabolically active organ functionally for about around 1-4 hours, it is clear that the technique which would be able to avoid deep hypothermia and still be able to preserve at least about 90% of normal function of organs after 24 hours post removal from the donor would be extremely advantageous.
The principles of successful preservation solution are a follows: energy conservation through the chemical induction of rapid and complete arrest of metabolic processes or at least slowing of metabolic rate to basal state and degradative processes through the coincident use of hypothermia and the prevention or reversal of certain unfavorable ischemia-induced changes with various protective agents.
Other attempts were made using various chemical means to achieve rapid offset of metabolic activity. Preservation solutions were investigated containing high concentrations of various ions such as potassium where infusion of a solution containing 16 mmol potassium chloride/litre caused decreased metabolism within a few seconds. The effect of this upon organ energy reserves and resistance to ischaemia has been investigated in a study in which isolated rat hearts were subjected to a 2 minute period of coronary infusion with a cardioplegic (16 mmol potassium/litre) or a non-cardioplegic (5 mmol potassium/litre) solution immediately following aortic cross clamping. After 30 minutes of ischaemia, the cardioplegic hearts contained 11.1 .+-.4.2 .mu.mol of ATP/g dry weight and 9.4.+-.2.1 .mu.mol of creatinine phosphate/g dry weight, whereas the corresponding figures in the non-cardioplegic group were 5.3.+-.0.9 and 2.8 .+-.0.4 .mu.mol/g dry weight respectively. This striking difference in high energy phosphates was reflected in the post-ischemic recovery of function, which was zero in the non-cardioplegic group as opposed to almost 50% in the cardioplegic group.
Potassium is not unique in its ability to induce decrease in metabolism. Numerous other agents have been used clinically and/or experimentally, for example zero calcium, high magnesium, acetylcholine, neostigmine, tetrodotoxin and other pharmaceutical agents. In each instance, the primary protective effect has been through rapid induction of metabolic arrest and conservation of cellular energy supplies. KCl over longer period of time then 2 hours has led to vascular constriction. In the light of current knowledge, however, some agents such as zero calcium or tetrodotoxin could not be recommended for clinical use, or in case of transplantation of the heart or other organs, since they can cause cellular activity inequilibrium.
Successful preservation of the organ for transplantation depends on maintenance or restoration of its full physiological function.
The most commonly used preservation solutions for transplantation include crystalloid cardioplegia consisting of isotonic or slightly hypertonic saline supplemented with glucose and potassium chloride of which buffering capacity is usually afforded by the addition of sodium bicarbonate or THAM. In addition, some solutions contain small amounts of magnesium or calcium, glucose, ATP and creatine phosphate, while others contain pharmacologic agents such as mannitol, insulin, procaine or calcium channel blockers. Blood cardioplegia as a preservation medium was also investigated but was not better than the other cardioplegia.
Despite these advances in development of these preservation solutions, a significant percentage of patients continue to demonstrate clinical evidence of organ damage in the postoperative period (New Engl. J. Med., 301:135 (1979), indicating that the current solutions for preservation of organs are not suitable for purposes of the organ preservation for transplantations for longer period of time.
For preservation of cellular mitochondrial function, it is important to arrest the organ metabolism immediately since significant utilization of high energy phosphates occurs during the brief period of organ's physiological activity between the onset of ischemia and the onset of a systole. J. Thorac. Cardiovasc. Surg., 77:803 (1979); J. Surg. Res., 24:201 (1978). This is particularly important since organ tissue recovery from prolonged global ischemia depends in part on the conservation of high energy phosphate stores and on the avoidance of reperfusion injury at the cellular level. J. Mol. Cell. Cardiol., 13:941 (1981).
Decrease in cardiac performance due to insufficient supply of free energy is well documented. A reduction in contractile performance of isolated hamster heart correlates with a decrease in free energy of ATP hydrolysis. Cardiac Adaptation to Hemodynamic Overload, Training and Stress., 197 (1983), Ed. R. Jacob et al., Steinkopff Verlag.
When the glucose was used as a sole substrate in isolated heart, the high energy phosphates ATP and phosphocreatine reached maximum values during heart diastole and minimum during systole. Upon exhaustion of ATP, a decrease in high level phosphate accompanied by a low level in the free energy of ATP hydrolysis, augmented levels of lactate and inorganic phosphate resulted in a 50% reduction of cardiac performance. Cir. Res., 53:759 (1983). These results are equally applicable for other organ although probably depending on amount of mechanical or metabolic activity the organ is performing.
Since during the normal function of the organ the high level of energy is required, and since when the organ is removed from the donor for the transplantation the supply of oxygen and nutrients is limited to those amounts present in the organ prior to its removal or to those present in the preservation solution, and since the primarily used energy substrate glucose is metabolized in the muscle cells to lactate, the organ tissue soon faces a metabolic acidosis. Under any kind, but in particular under high work-load, conditions with glucose as a sole substrate, glycolytic production of pyruvate is inadequate to meet the energy needs under aerobic or anaerobic conditions, and consequently tissue, edema and acidosis develops. The acidosis, in turn may have a number of detrimental effects on organ function during disruption of circulation including cellular abnormalities and a reduction in functional performance, and gradual change in cellular homeostasis. Moreover, incomplete oxidation of glucose and the resultant increase in sugar phosphate is known to cause the cell edema which is very undesirable condition for the tissue preservation. Consequently, a suitable substitute for glucose would be advantageous.
Without the energy and nutrients supply, the metabolic depletion of intracellular ATP impairs the chance for post ischemic or post metabolic deficiency recovery of the organ performance. Thus, the same kind of energy providing substrate is necessary to be present in the preservation solution.
If glycolysis is rate limiting, there is reduced delivery of pyruvate to the intracellular mitochondria. By substituting pyruvate for loading the cells with glucose, glycolysis is bypassed and pyruvate is available to the mitochondria for oxidative phosphorylation producing free energy ATP. Ann. J Physiol., 253: H 1261 (1987). Moreover, pyruvate does not cause cell edema. The use of pyruvate as a sole exogenous substrate results in greater functional and biochemical recovery.
Circ. Res., 35:448 (1974) reports that intracellular Ca.sup.2+ -overload leads to impaired oxidative phosphorylation, increased ATP breakdown and consequently inefficient ATP utilization for mechanical work.
Using the currently available organ preservation cardioplegic solutions, the safe time for organ survival is from 30 minutes to about 4 hours in human. As reported in Heart Disease, 1962 (1988), 3rd Ed., Harcourt Brace Jovanovich, within this period no significant tissue necrosis or permanent functional damage results. Under these conditions, Principles in Surgery, 407 (1984) 4th Ed., McGraw Hill, suggest that majority organs for transplantation in humans should be optimally implanted within 4 hours from the time of the organ removal.
Therefore, it would be advantageous for a world-wide transplantation network to extend this time period to possibly 24 hours or more. With the transportation feasibility to connect around the world in 24 hours, a supply of the various organs for transplantation could be widely improved and made practical if these organs were able to be fully functional after 24 hours.
It has been previously reported, that the metabolism of the organs is severely affected by ingestion of large amounts of alcohol. Changes such as impaired sodium, potassium stimulated ATPase activity, inhibition of sodium-calcium exchange, decreased fatty acid oxidation, depressed ATP, impairment of mitochondrial function and diminished ratios of phosphate to oxygen all lead to a reduction in organ function. While higher alcohol concentrations have been reported to produce a sudden cardiac arrest in the isolated hamster and rat heart, acute alcohol exposure reversibly depresses cardiac function without affecting energy resources. FASEB. J., 2:256 (1988) and Mag. Res. in Med., 8:58 (1988) reported that perfusion of the isolated hamster heart with 2% ethanol for 30 minutes showed decrease in developed pressure, a marked increase in enddiastolic pressure, a decrease in ATP and an increase in inorganic phosphate. There was no change in phosphocreatine or intracellular pH. After reequilibration, all the above values returned to almost normal levels showing that alcohol induced functional metabolic depression is reversible.
It is a primary object of this invention to provide an improved technology for long-time preservation of the organs for transplantation by using novel preservation solutions and process of using these solutions to achieve the almost complete functional restoration of isolated organs after 24 hours ischemia.